Materials and methods for detection and treatment of immune system dysfunctions

ABSTRACT

The subject invention concerns novel materials and methods for the treatment and/or prevention of autoimmune disease. In a specific embodiment, elevated production of prostaglandin synthase-2 (PGS-2) is correlated with autoimmune dysfunction.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of application Ser. No.09/322,628, filed May 28, 1999; which is a division of application Ser.No. 08/916,586, filed Aug. 22, 1997, now U.S. Pat. No. 6,168,792; whichis a continuation-in-part of application Ser. No. 08/701,928, filed Aug.23, 1996, now U.S. Pat. No. 5,939,069.

BACKGROUND OF THE INVENTION

[0002] Diabetes is a term that refers to a collection of diseasesresulting in disordered energy metabolism and varying degrees of bloodglucose elevations or hyperglycemia. One of the best characterized formsof the disease is that which arises from an immunologically mediateddestruction of the insulin secreting pancreatic beta cells. This severeform of the disease is termed Insulin-dependent Diabetes (IDD or IMD)since it is associated with progressive insulin deficiency andcoincident symptoms such as weight loss, glycosuria and polyuria, andincreased thirst or polydipsia. Other terms for this form of diabetesare Type 1 Diabetes (cf. Type 2 Diabetes which results from an inherentresistance to insulin action); Ketosis Prone Diabetes because there isabnormal generation of ketone bodies as a result of excessive breakdownof body fats due to the severe insulin deficiency; or Juvenile Diabetes,since virtually all diabetes that appears in childhood and adolescenceis of this type.

[0003] Diabetes is a major public health problem, especially in Westerncountries. The incidence rates vary greatly worldwide, from as high as40 per 100,000 persons in Finland to as low as 1-2 per 100,000 among theJapanese. The peak incidence is during the pubertal years, associatedwith the increasing bodily demands for insulin associated with musclegrowth. The prevalence rates in the United States population under age20 years is 0.25% and it approaches 0.4% over a lifetime, albeit anestimated 10-20% of patients with Non Insulin-dependent Diabetes (NIDD)or Type 2 or Maturity Onset Diabetes also have, in reality, slowlyprogressive IDD. Thus, it is estimated that there may be at least 1million Americans affected by IDD.

[0004] Diabetes results in progressive damage to the blood vessels ofthe body, to a degree that depends upon the severity of hyperglycemiaand its duration. The incident mortality rate for IDD has beencalculated to be 7-fold higher than for age matched non-diabeticcontrols. Whereas the decade long Diabetes Control and ComplicationsTrial (DCCT)—concluded in 1994 by the National Institutes of Health inthe United States—showed that meticulous insulin replacement therapywould slow the appearance of damaged arteries, it was not able tocompletely prevent this damage since blood glucose levels were difficultto keep within normal limits. Ocular complications of diabetes are theleading cause of new blindness in persons 20-74 years of age. The riskof lower extremity amputation is 15-fold higher in those with diabetes.Approximately 40% of persons undergoing renal transplantations havekidney failure because of diabetes, and the proportion due to diabetescontinues to rise each year. Women with diabetes produce newborn infantswith a 7% newborn mortality rate. Other complications of diabetesinclude increased heart disease and stroke, loss of nerve cells orneurons enervating the limbs and intestine, impotence and infertility,cataract formation in the lens of the eyes, increased periodontaldisease, and predisposition to infectious diseases especially frombacteria and yeast. Of all patients with diabetes, those with IDD have adisproportionate share of these complications because of its severityand usual early age of onset. In the United States, the direct healthcare costs attributable to diabetes in 1994 have been estimated toexceed $120 billion. Thus it is important that the pathogenesis of IDDbe understood and strategies be developed to prevent it as a fullyexpressed clinical disease.

[0005] Patients with IDD are unusually prone to other diseases that havebecome recognized as having autoimmune origins. These diseases includethyroiditis or Hashimoto's disease, Graves' disease, Addison's disease,atrophic gastritis and pernicious anemia, celiac disease, and vitiligo(Maclaren, N. K. [1985] Diabetes Care 8(suppl.):34-38). Evidence thatIDD itself has an autoimmune nature began with histological studies ofpatients; these studies indicated that the islets were infiltrated witha chronic inflammatory (lymphocytic) infiltrate termed insulitis. Thiswas supported in the early 1970s by reports of islet cell autoantibodiesreactive to antigens within the cytoplasm (ICA) (Lendrum et al. [1975]Lancet 1:880-882) or confined to the islet cell surfaces (ICSA)(Maclaren et al. [1975] Lancet 1:977-1000) as detectable by indirectimmunofluorescence. Later it was recognized that many patients alsodevelop autoantibodies to insulin (IAA) before their diagnosis (Palmeret al. [1983] Science 222:1337-1339) as well as to insulin receptors(Maron et al. [1983] Nature 303:817-818). Autoantibodies were alsoreported to an islet cell protein composition of 64,000 M.Wt. in man(Baekkeskov et al. [1982] Nature 298:167-169), in the Biobreeding (BB)rat model (Baekkeskov et al. [1984] Science 224:1348-1350), and in theNon Obese Diabetic (NOD) mouse model (Atkinson and Maclaren [1988]Diabetes 37:1587-1590). 64 kDa antigen has subsequently been reported tobe the lower molecular weight isoform of glutamic acid decarboxylase(GAD₆₅) (Baekkeskov et al. [1990] Nature 347:151-156) (Kauffman et al.[1992] J. Clin. Invest. 283-292). GAD is an enzyme that convertsglutamate into the membrane stabilizing neurotransmitter called gammaamino butyric acid or GABA. In addition to autoantibodies to GAD,peripheral blood mononuclear cells were shown to be autoreactive inpatients developing IDD (Atkinson and Maclaren et al. [1992] Lancet339:458-459; and Harrison et al. [1993] Lancet 341:1365-1369).

[0006] It has previously been demonstrated in several autoimmunediseases, including IDD, systemic lupus erythematosus (SLE), rheumatoidarthritis (RA), multiple sclerosis (MS), and autoimmune thyroid disease,that antigen-presenting cells (APCs) such as monocytes and macrophagesare dysfunctional in their ability to activate T lymphocytes (Via, C. S.et al. [1993] J. Immunol. 151:3914-3922; Serreze, D. [1993] FASEB J.7:1092-1096; Rasanen, L. et al. [1988] Clin. Exp. Immunol. 71:470-474;Hafler, D. A., et al. [1985] J. Neuroimmunol. 9:339-347). The defect(s)in APC function, however, have thus far not been defined at the cellularor molecular level.

[0007] Prostaglandins (PGs) are lipid molecules formed from a precursormolecule, arachidonic acid, through the actions of specific enzymescalled prostaglandin synthases (PGS-1 and PGS-2). PGS-1 mRNA and proteinare constitutively expressed, and this enzyme is responsible for theproduction of low levels of PGs and functions as a housekeepingmolecule. PGS-2 is an inducible enzyme expressed by macrophages andmonocytes during inflammation and following exposure to mitogens,cytokines, and bacterial cell wall products, i.e., lipopolysaccharide(LPS) (Farber, J. M. [1992] Mol. Cell. Biol. 12:1535-1545; Vane, J. R.[1994] Proc. Natl. Acad. Sci. USA 91:2046-2050; Kujubn, D. A. [1993] J.Biol. Chem. 266:12866-12872; Ristimaki, A. et al. [1994] J. Biol. Chem.269:11769-11775). PGS-2 has been shown to be expressed in the cellslining the joints of individuals with rheumatoid arthritis and maycontribute to the ongoing inflammation in the affected joint (Crofford,L. J. et al. [1994] J. Clin. Invest. 93:1095-1101).

[0008] In addition to prostanoids, monocytes produce monokines thatstrongly modify inflammation and immune responses. Among the monokinesthat in general upregulate or promote inflammation and immunity areTNFα, IL-1α, IL-1β, IL-12, and IL-6. Monokines that tend to downregulatethese responses are IL-4, IL-10, IL-13, and IL-1 receptor antagonist(IL-1RA).

[0009] Prostaglandins are known to have an effect on the expression ofmonokines. For instance, prostaglandin E₂ (PGE₂) is known to suppressthe production of TNFα (Seldon, P. M. et al. [1995] Mol. Pharmacol.48:747-757; Strieter, R. M. et al. [1990] J. Leuk. Biol. 47:366-370).There are also reports that PGE₂ suppresses IL-1α (Endres, S. et al.[1991] Immunology 72:56-60; Zhong, W. W. [1995] Immunology 84:446-452).In contrast to its suppressive effects, PGE₂ production stimulates theproduction of IL-10 by monocytes (Strassman, G. et al. [1994] J. Exp.Med. 180:2365-2370). IL-10 in turn modulates PGE₂ production bysuppressing PGS-2 production (Mertz, P. M. et al. [1994] J. Biol. Chem.269:21322-21329). In addition, IL-10 stimulates the production ofanother potent immunoregulatory monokine, IL-1RA (Spengler, R. N. et al.[1989] J. Immunol. 142:4346-4350). PGE₂ therefore can stimulateimmunoregulation through its own action and through its actions onmonokine production.

[0010] If monocytes are chronically exposed to PGE₂ in vitro, there is aloss of response to its action. This desensitization phenomena ismediated by the down-regulation of PGE₂ receptors (Coffey, R. G. et al.[1990] J. Leuk. Biol. 48:557-564). For example, PGE₂ in vitro normallysuppresses TNFα production, but chronic exposure leads to a loss ofsuppression of this monokine by PGE₂. Removal of, or blocking, PGE₂ canreverse the desensitization process (Howard, M. et al. [1992] J. Clin.Immunol. 12:61-784).

[0011] Currently, one can screen for individuals at high risk for thedevelopment of IDD by serologic methods only, which reflect autoimmune Band T lymphocyte activity. Serologic tests identify approximately 80-85%of individuals who have existing autoimmune disease against theinsulin-producing cells of the pancreas. Of the ICA+ population,approximately 80-85% will develop IDD within the ensuing five years.Currently no test exists, immunologic, genetic, or otherwise, which canidentify individuals at risk for IDD other than these serologic tests.

[0012] Because IDD takes several years to develop in an individual,autoimmunity may be firmly established at the time that individualsdevelop ICAs. Detection of background cellular or genetic factorsnecessary for the development of autoimmune disease and expressed earlyin the disease process is of great clinical importance. Detection ofthese factors would preferably identify individuals before autoimmunityis initiated, or perhaps at earlier stages of the disease than detectedby ICA. Earlier detection would be of great clinical importance inidentifying individuals at high risk for disease where theadministration of preventative therapies that attempt to preserve theresidual insulin-secreting cells are employed.

BRIEF SUMMARY OF THE INVENTION

[0013] The subject invention pertains to materials and methods for thedetection, prevention, and treatment of diabetes, other autoimmuneconditions, and conditions involving dysfunctional apoptotic processes.In a specific embodiment, the subject invention concerns theidentification of a defect in antigen-presenting cells (APCs) that isassociated with diabetes and other autoimmune disorders. One aspect ofthis defect is the elevated production of prostaglandin synthase-2(PGS-2) in cells of individuals who have developed or will developautoimmune disease.

[0014] In a specific embodiment of the subject invention, diabetesand/or other autoimmune disease can be predicted and/or monitored byassaying for expression of prostaglandin synthase-2 (PGS-2) byantigen-presenting cells. Thus, one aspect of the invention is thediscovery that PGS-2 is a cellular marker that is strongly associatedwith clinical autoimmune diseases such as IDD. In a preferredembodiment, the antigen-presenting cells which are monitored fordiagnostic purposes are macrophages and/or monocytes. Expression ofPGS-2 in these cells is an indicator of autoimmune susceptibility ordisease.

[0015] The expression of PGS-2 can be detected in any of a number ofways which would be apparent to those skilled in the art having thebenefit of this disclosure. For example, the expression of PGS-2 can bedetected by the presence of PGS-2 messenger RNA (mRNA), presence of thePGS-2 protein itself, or by detecting biological effects of the PGS-2protein, i.e., PGE₂ production.

[0016] A further aspect of the subject invention pertains to theidentification of diagnostic markers for autoimmune disease on T-cells.In one embodiment it has been found that CD25 expression on T-cells canbe correlated with autoimmune disease such as IDD. Specifically, it hasbeen found that individuals with a susceptibility to diabetes have alower level of expression of CD25 on T-cells. Furthermore, inhibition ofPGE₂ was found to significantly increase CD25 expression in cells ofindividuals at risk for IDD. Inhibition of PGE₂ did not enhanceexpression of CD25 in individuals who are not at risk to develop IDD. Ina further embodiment, individuals at risk for IDD have been found tohave decreased levels of FAS receptor expression compared to individualswho are not at risk to develop IDD. In a preferred embodiment T-cellswhich express both FAS receptor and CD25 are examined for a reducedlevel of expression of these proteins. A reduced, level of expression ofFAS receptor and CD25 compared to normal controls is indicative ofdiabetes or other autoimmune conditions.

[0017] Assays for PGS-2 expression, CD25 expression and/or FAS receptorexpression add new dimensions to disease prediction such as assessingdisease activity and progression, and predisposition for developingother autoimmune diseases.

[0018] The diagnostic procedures described herein can be used to detectevidence of autoimmune dysfunction before the appearance of clinicalsymptoms. This early detection makes it possible to initiate appropriatepreventative measures.

[0019] A further aspect of the subject invention is the discovery thatpharmacologic inhibition of PGS-2 has potent inhibitory effects on thedevelopment of autoimmune disease. In one embodiment, prevention of IDDcan be achieved according to the subject invention by drug therapy thatblocks PGS-2 enzymatic activity. PGS-2 inhibitors provide aninexpensive, safe, and well-tolerated approach to the prevention of thisdisease. Furthermore, the efficacy of therapy can be monitored bymeasuring serum or urine PGE₂ levels. Also, for certain individuals,PGS-2-specific inhibitors were found to markedly reduce production ofPGE₂ and increase IL-1 receptor antagonist (IL-1RA) in human monocytes.

[0020] A further aspect of the subject invention pertains to treatmentsfor diabetes or other autoimmune disease which comprise modulating CD25expression and/or FAS receptor expression. In a preferred embodiment,individuals at risk for IDD are treated to increase CD25 expression.This increase can be effected by, for example, administering a compoundwhich inhibits PGS-2 activity, PGE₂ activity, or the activity of cyclicAMP or related compounds. Such inhibition can be achieved by, forexample, an inhibitor of PGS-1 or PGS-2. In a preferred embodiment, aPGS-2 specific inhibitor such as NS398 is administered.

[0021] A further aspect of the subject invention pertains to diagnosticand therapeutic methods based on modulation and/or detection of celldeath mechanisms and events. In accordance with the subject invention,differences in cell death events relating to expression or activity ofPGS-2 and/or related molecules can be exploited to provide criticaldiagnostic information or to intervene in disease processes. In aspecific embodiment, individuals at risk for cancer or to developautoimmune diseases will display a PGS-2 related resistance to celldeath upon stimulation of cells by chemical factors including, but notlimited to, TNFα and FAS ligand. By intervening in this process by, forexample, the administration of inhibitors of PGS-2-related activity, itis possible to facilitate the completion of cell death events toeliminate inappropriate cells from the biological milieu. In this way,autoimmune T-cells can be removed through the apoptic mechanism uponstimulation by appropriate chemical signals or immunization withdisease-related target antigens such as insulin or GAD. Similarly,cancer cells can also proceed to appropriate cell death, therebypreventing or reducing tumors and/or other inappropriate cellularproliferation.

[0022] Thus, a further specific embodiment of the subject inventionconcerns the administration of inhibitors of PGS-2, or its biologicalactivities, to effect a modulation of programmed cell death such thatself-destructive T-cells and/or cancer cells are removed to reduce orprevent autoimmune or cancer conditions. The inhibitors of PGS-2 whichcan be used according to the subject invention include, but are notlimited to, glucocorticoid hormones (which suppress expression ofPGS-2), IL-10, IL-4, IL-13, and TGF-β.

[0023] In a further embodiment of the subject invention modulation ofcell death can be achieved by upregulating the cellular responseresulting from activation of the FAS receptor. This upregulation can beachieved by administration of an agent which increases FAS receptorexpression. This agent may be, for example, a PGE inhibitor.Alternatively, the upregulation of the FAS cellular response can beachieved by, for example, administration of agents which enhance theintracellular response to FAS receptor activation. Thus, in individualshaving a pathological condition attributable to aberrant cell deathprocesses, cell death can be promoted by upregulating the intracellularcascade of events whereby FAS receptor activation ultimately promotescell death. This upregulation can be achieved by those skilled in theart, having benefit of the instant disclosure, by, for example,stimulating enzymatic and other regulatory molecules which participatein the FAS activation pathway. In a specific embodiment, individualsidentified as needing increased T-cell death can be treated to increaseboth CD25 expression and cellular response to FAS receptor activation.This therapy can be further augmented by administration of anappropriate antigen thereby enhancing CD25 activation and increasing thespecificity of the treatment. The antigen may be, for example, anautoantigen.

BRIEF DESCRIPTION OF THE DRAWING

[0024]FIG. 1 shows the blocking of PGE₂ production by the PGS-2-specificinhibitor, NS-398.

[0025]FIG. 2 shows the correlation between increased PGS-2 levels and anincreased risk of IDD.

[0026]FIG. 3 shows the ability of a PGS-2 inhibitor to increaseexpression of CD25.

DETAILED DISCLOSURE OF THE INVENTION

[0027] In one aspect the subject invention pertains to the detectionand/or modulation of prostaglandin synthase-2 (PGS-2) production inantigen-presenting cells (APCs) of individuals at risk for developingautoimmune disease or cancer. In this embodiment, the subject inventionconcerns the discovery that PGS-2 is a cellular marker for individualsat risk for IDD and other autoimmune diseases. Another embodiment of thesubject invention concerns preventative or therapeutic treatments. In apreferred embodiment, drugs that block PGS-2 production or activity canbe administered to individuals who have autoimmune disease or cellularproliferation disease, or are at risk for developing such diseases. In afurther embodiment, the subject invention pertains to monitoring and/ormodulation of CD25 expression and/or FAS receptor activation of T-cellsor other cells having dysfunctional cell death processes.

[0028] The subject invention provides quick and easy procedures fordetermining whether an individual may be at risk for developingautoimmune disease. In one diagnostic aspect of the invention,peripheral blood monocytes can be evaluated to determine the level ofPGS-2 expression. This evaluation can be conducted using any one of anumber of diagnostic procedures well known to those skilled in the art.These procedures may be used to detect PGS-2 directly or to detectevidence of PGS-2 expression. Evidence of PGS-2 expression includes, forexample, the presence of PGS-2 mRNA. The PGS-2 mRNA can be detected by,for example, reverse transcriptase PCR (RT-PCR). PGE₂, which is producedon account of the enzymatic activity of PGS-2, can readily be detectedusing, for example, ELISA, RIA, or other antibody-based assays. Theexpression of PGS-2 protein can also be detected using flow cytometrymethods or Western blotting.

[0029] In a further diagnostic aspect of the subject invention, T-cellsare evaluated to determine levels of CD25 expression and/or FAS receptorexpression. A decrease in expression of either CD25 or FAS receptor hasbeen found to be indicative of risk for autoimmune disease—particularlyIDD. A decrease in the combined expression of CD25 and FAS receptor isparticularly indicative of risk for autoimmune disease. The level ofexpression of these molecules can be readily ascertained by thoseskilled in the art using standard techniques such as, for example, FACSanalysis using commercially available antibodies. A further diagnosticprocedure involves the determination of the increase in CD25 expressionresulting from the administration of a PGE-2 inhibitor. An increase inCD25 expression on T-cells upon treatment with a PGE-2 inhibitor isindicative of risk for autoimmune disease including IDD. The PGE-2inhibitor may be, for example, NS398 or indomethacin, which inhibitsboth PGS-1 and PGS-2.

[0030] The diagnostic assays of the subject invention can be used todetect evidence of autoimmune disease before the appearance of clinicalsystems. Furthermore, the assays are useful for monitoring diseaseprogression or the effect of treatment.

[0031] In a therapeutic and/or preventative aspect of the subjectinvention, the action of prostaglandins can be blocked or inhibited,thereby slowing or eliminating undesirable immune processes. In apreferred embodiment, the activity of PGS-2 and/or related molecules canbe blocked. Indomethacin is known to block the activity of PGS-2.Aminoguanidine is an inhibitor of inducible nitric oxide synthase(iNOS). NO augments PGS-2 activity. Thus, the subject invention providesa method for reducing the severity of autoimmune disease. As used hereinreference to “reducing the severity” of a disorder would includepreventing or delaying the disorder or making the effects of thedisorder less damaging physically or emotionally.

[0032] Treatment of NOD mice afflicted with active, establishedautoimmune disease using a drug combination of indomethacin andaminoguanidine markedly delays the onset and reduces the incidence ofdiabetes. The combination of inhibitors is particularly effective.Furthermore, treatment of NOD mice at an early stage of disease withindomethacin alone effectively blocks the development of IDD. It hasalso been found that the PGS-2-specific inhibitor, NS-398 (availablefrom Cayman Chemical Company), effectively blocks all PGS-2 productionin vitro from monocytes of individuals at high risk for IDD. Treatmentof individuals at high risk for IDD can be used to block the progressionof autoimmunity to frank diabetes.

[0033] The treatments of the subject invention are also useful to reducethe severity of the side effects of autoimmune disease. In aparticularly preferred embodiment the side effects of diabetes can bereduced by treatment with a prostaglandin inhibitor.

[0034] In a further embodiment of the subject invention, PGS-2-specificinhibitors can be used in conjunction with antigen-specific immunizationtherapy. In the immunization treatment paradigms, a target antigen forIDD, or other autoimmune disease, is used to immunize the individual.This results in tolerance and a lack of progression to overt disease. Inthe case of diabetes, antigens useful in this regard include, but arenot limited to, insulin, GAD, IA-2, IA-2β, and fragments and variants ofthese antigens. Antigens associated with various autoimmune conditionsinclude, but are not limited to, those shown below in Table 1. TABLE 1Condition Antigens Multiple Sclerosis myelin basic protein proteolipidprotein Rheumatoid Arthritis collagen Lupus DNA histone proteins IDDMGAD insulin IA-2 IA-2β 38 kD protein perforin Thyroid thyroglobulinperoxidase Vitiligo tyrosinase

[0035] As PGs inhibit lymphocyte activation, and activation is aprerequisite for apoptosis of T cells, treatment of subjects with PGS-2inhibitors prior to, or concurrent with, immunization can potentiate theeffects of this therapy. Therefore, PGS-2 inhibitors can be used as anadjuvant therapy for antigen immunization to prevent IDD or otherautoimmune diseases.

[0036] The monocyte production of the monokines IL-1β, TNFα, IL-1RA, andIL-10 in culture supernatants from healthy controls and the pre-diabeticpopulation in the presence and absence of the PGS-2-specific inhibitor,NS-398, have been evaluated. It has been determined that NS-398 promotesthe production of the immunoregulatory monokines IL-10 and IL-1RA,suggesting that high levels of PGE₂ produced by pre-diabetic monocytesresult in PGE₂ desensitization. It has also been determined that,despite enhanced levels of PGS-2 and PGE₂ in diabetic and pre-diabeticindividuals, there is no corresponding increase in IL-10. Furthermore,in NOD mice, administration of IL-10 surprisingly does not inhibit PGE₂production by monocytes. Therefore, a further aspect of the subjectinvention is a method for detecting evidence of autoimmune disease,particularly diabetes, which comprises evaluating monocytes to determineif those monocytes have a reduced response to IL-10 with regard to PGS-2expression. Such reduced response would be evidence of disease. Inaccordance with these findings, compounds that inhibit prostaglandinproduction can be used to modulate the production of anti-inflammatorymonokines in order to limit the immune response. The spontaneousexpression of PGS-2 and the production of PGE₂ by pre-diabetic monocytesmay influence their function, limiting their ability to producemonokines that would have a potent effect on limiting the autoimmuneresponse to the β-cells of the islet and therefore would promote theprogression to diabetes.

[0037] Treatments that inhibit PGE₂ can be used according to the subjectinvention to restore the production of these potent regulatory monokinesand either slow or block the autoimmune process, as well as to promoteappropriate apoptotic processes. In addition, the production of IL-10 bythe monocyte can be used to promote the production of Th2 lymphocytesthat are thought to play an important regulatory role in IDD.Furthermore, blocking PGE₂ and limiting prostaglandin desensitizationcan also be used to promote the generation of TH2 cells as PGE₂ promotesthe generation of these cells.

[0038] A further therapeutic embodiment of the subject inventionpertains to the modulation of CD25 expression of T-cells. In a relatedaspect of the subject invention the cellular response to FAS receptoractivation can be modulated. For individuals at risk for IDD or in needof increased programmed cell death, the subject invention provides atreatment whereby the efficiency of the cell death pathway is enhanced.This enhancement is accomplished by increasing expression of CD25 and/orthe response to FAS receptor activation. In a preferred embodiment, theenhancement of CD25 expression can be accomplished by administering aninhibitor of PGE₂. Upregulation of FAS-related cellular activation ispreferably achieved by increasing expression of the FAS protein or byenhancing the amount, or activity, of compounds which promote theintracellular FAS activation pathway. In one embodiment, this therapy isaugmented by the administration of an antigen thereby enhancing theactivity resulting from expression of CD25. The antigen may be, forexample, an autoantigen.

[0039] In a specific embodiment, the subject invention comprisesadministering a prostaglandin inhibitor to an individual who has beendetermined to be at risk for developing autoimmune disease. In apreferred embodiment, the prostaglandin inhibitor is administeredchronically. In a further preferred embodiment, the prostaglandininhibitor is administered at a dose which is higher than that whichwould be used, for example, to relieve pain or inflamation. In thisregard, the inhibitor should be administered at a dosage and in a mannerwhich will effectively increase expression of CD25 on T-cells. In aparticularly preferred embodiment, the prostaglandin inhibitor is aninhibitor of PGS-2. The therapy of the subject invention is particularlyadvantageous when administered prior to the appearance of clinicalsymptoms of autoimmune disease or the need for enhanced programmed celldeath.

Materials and Methods

[0040] RT-PCR detection of PGS-2 mRNA in human monocytes. Humanperipheral blood is collected in sterile green top 10 ml tubes. Assaysare preferably performed with 5 ml or more of blood. The blood is thencentrifuged on a ficoll gradient for 30 minutes at 1500 rpm. Monocytesare isolated to >80% purity by adherence for 2 hours to a plasticculture surface. Cells are then cultured overnight in RMPI-1640 plusendotoxin-free fetal calf sera. The cells are harvested after 16 hoursof culture by cold Ca⁺⁺/Mg⁺⁺-free PBS, the cells are counted, andviability is assessed. Monocytes cultured in 10 μg/ml LPS serve as apositive control for each sample tested. The poly mRNA is then harvestedfrom a standard 10⁵ monocytes using a kit (Quiagen). The mRNA is thenreverse transcribed in a standard reaction mixture. The cDNA is thenamplified by PGS-2-specific primers designed by our laboratory using astandard reverse transcriptase reaction for 45 cycles. The PCR productis then run out on agarose gels with known standards and the sizeconfirmed. β-actin is used as an internal control in these reactions.The identity of RT-PCR products were confirmed by size and Southernblotting using a PGS-2-specific labeled internal probe.

[0041] PGS-2 protein detection in human monocytes. A PGS-2-specificmouse monoclonal antibody (Cayman Chemical Company) has been utilizedfor detection of PGS-2 protein in monocytes. Cells forimmunocytochemistry are adhered to multichamber slides for two hours,fixed in 0.2% paraformaldehyde, and permeabilized with Triton-X andglycine in PBS for 10 minutes. The primary antibody is incubatedovernight at 4° C. and then detected with FITC labeled Fab goatanti-rabbit antisera. The cells are then visualized with a fluorescentmicroscope.

[0042] A fluorescent activated cell sorting (FACs) method can also beused for detection of PGS-2 in monocytes. In this procedure, whole bloodis labeled with an anti-monocyte antibody CD14 coupled to aphycoerythrin molecule. The cells are then fixed and lysed withFACs-Lyse (Becton-Dickenson). The cells are then further permeabilizedwith saponins throughout the procedure, and then analyzed on a FACsmachine. The percent of monocytes positive for PGS-2, as well asfluorescent intensity, is then determined. Approximately 15%-70% ofperipheral blood monocytes from ICA+ individuals are positive by thismethod.

[0043] Following are examples which illustrate procedures for practicingthe invention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Detection of PGS-2 mRNA

[0044] The regulation of mRNA and protein expression of PGS-2, theinducible enzyme critical for the production of large quantities ofprostaglandins, was examined in macrophages of the NOD mouse.Macrophages from control mouse strains did not express PGS-2 mRNA asdetermined by the highly sensitive reverse transcriptase polymerasechain reaction. The PGS-2 protein is likewise not expressed in restingcontrol macrophages as assessed by indirect immunofluorescence using aPGS-2 specific antibody. In marked contrast, NOD macrophagesspontaneously express high levels of mRNA and protein for this enzyme asdetermined by these techniques. PGS-2 was also found to be expressed inthe macrophages of NODscid/scid mice which lack functional T and B cellsand, as a result, do not develop autoimmune disease or diabetes. Theexpression of PGS-2 in macrophages of NODscid/scid mice indicates thatspontaneous PGS-2 expression is not dependent on the autoimmune milieu,and suggests that PGS-2 expression is a result of a primary macrophagedefect. Therefore, the aberrant expression of PGS-2 by NOD macrophagesreadily explains the abnormal PG production by these cells and providesthe molecular and cellular basis for APC dysfunction.

EXAMPLE 2 Role of PGS-2 Expression in IDD

[0045] In order to more fully establish the genetic contribution ofPGS-2 expression to autoimmunity in the NOD mouse, congenic mice wereexamined. The gene encoding PGS-2 is located on chromosome 1, 76.2 CMfrom the centromere. These mice, designated B6.NOD.C1, have a segment ofchromosome 1 from the NOD mouse that contains the “NOD PGS-2 gene,”while the rest of the mouse genome is from the non-autoimmune C57BL/6mouse. Macrophages from B6.BOD.C1 congenic mice, like the NOD,spontaneously express PGS-2 mRNA and protein. These mice developautoimmune disease in the pancreas, but unlike the NOD, do not developdiabetes. The absence of diabetes in these congenic mice is likely dueto a lack of other key genes from the NOD that contribute otherimportant factors to the overall disease process. Another line ofcongenic mice were designated NOD.B10.C1. These mice contain the NODgenome except for a segment of chromosome 1 derived from thenon-autoimmune C57BL/10 mouse and contains the “C57BL/10 PGS-2 gene.”NOD.B10.C1 mice do not spontaneously express PGS-2 and have a 40-50%reduction in the incidence of diabetes. These data, along with thosefrom the B6.NOD.C1 congenic mice suggest that the expression of PGS-2correlates with a more aggressive autoimmune phenotype.

EXAMPLE 3 Blocking Activity of PGS-2 as Therapy for IDD

[0046] Blocking the activity of PGS-2 with drugs that reduce PGproduction can be used to prevent or slow the development of IDDdisease. When NOD mice with established, active autoimmune disease aretreated through their drinking water with a combination of drugs,including a PGS-1/PGS-2 inhibitor, low dose indomethacin (3 μg/ml), inconjunction with an inducible nitric oxide synthase (iNOS) inhibitor,aminoguanidine, which potentiates the effects of indomethacin, theincidence of diabetes in NOD mice drops by 42% in comparison to controlanimals and animals treated with either low dose indomethacin oraminoguanidine alone.

[0047] Treatment of NOD mice at a time when autoimmunity is in its finalstages with high doses (15 μg/ml) of indomethacin alone reduces theincidence of diabetes from 77% to 22%. These data demonstrate a strongeffect of PGS inhibitors on the development of IDD.

EXAMPLE 4 Association of PGS-2 and PGE₂ with Autoimmune Disease

[0048] The effects of non-steroidal anti-inflammatory drugs (NSAIDs)that specifically inhibit PGS-2 in vitro were assessed, and they werefound to be highly potent blockers of NOD mouse macrophage PGS-2production. Aspirin-like drugs that inhibit both PGS-1 and PGS-2 causegastric irritation, whereas PGS-2-specific drugs do not.

[0049] Monocytes were examined from ICA+ humans with a high risk ofdeveloping IDD, individuals with established SLE and autoimmune thyroiddisease, and healthy controls. A portion of the pre-diabetic ICA+individuals examined are enrolled in an IDD trial where they receivedaily subcutaneous insulin as a preventative therapy. Monocytes ofhumans without autoimmune disease infrequently express PGS-2 mRNA (12%),whereas monocytes from subjects who are at high risk for the developmentof IDD express PGS-2 at a highly significant frequency (84%, p<0.0001).The preventative insulin therapy does not appear to affect PGS-2 mRNA orprotein expression (see Table 2). TABLE 2 PGS-2 mRNA expression in MOsof healthy controls and ICA⁺ subjects N PGS-2 + % Positive Healthycontrols 25  3 12% Females 12  0  0% Males 13  3 23% ICA⁺ 31 26 84%*Females 17 12 80% Males 14 11 85% Insulin RX 14 10 71% No RX 17 16 94%

[0050] PGS-2 expression, however, is not specific for IDD, as humanswith SLE and autoimmune thyroid disease also spontaneously express PGS-2in their monocytes (see Table 3). TABLE 3 PGS-2 expression in MOs ofautoimmune controls N PGS-2 + % Positive SLE 5 4 80% Hashimoto's 4 2 50%

[0051] In addition to the expression of PGS-2, the production of PGE2 bymonocytes from these same control and autoimmune individuals wasexamined. It was found that PGE2 production, as determined by specificELISA, is significantly higher in ICA+ individuals and in autoimmunecontrols than in control monocytes (p<0.0001) (see Table 4). TABLE 4PGE₂ production by MOs of healthy controls, ICA⁺, and SLE subjects NPGE₂ pg/ml Healthy controls 18  789 +/− 243* ICA⁺/PGS-2 (+) 23  7705 +/−1510* ICA⁺/PGS-2 (−)  5  288 +/− 211 ICA⁺/PGS-2 + /Ins. RX 10  5805 +/−1544 SLE/PGS-2 +  4 15437 +/− 12900

[0052] Finally, the production of PGE2 was completely blocked by thePGS-2-specific inhibitor NS-398 (see FIG. 1).

EXAMPLE 5 Monocyte Production of TNFα and IL-1β

[0053] The expression of PGS-2 can be induced by several factorsincluding cytokines produced by monocytes such as TNFα and IL-1β. Toevaluate the possibility that monocytes of pre-IDD subjects producelarge quantities of IL-1 and TNFα which induces PGS-2, the levels ofthese cytokines were measured in supernatants of monocytes cultured invitro for 24 hours from pre-IDD and healthy controls. As summarized inthe table below, it was found that the levels of both IL-1 and TNFαproduced by monocytes were actually lower in the pre-IDD subjects thanin the controls (see Table 5). The lower levels of these cytokines isconsistent with the constitutive production of prostaglandins whichsuppress the production of both TNFα and IL-1β. A comparison of TNFα andIL-1β levels between insulin-treated and untreated pre-IDD yielded nosignificant differences between these two groups. TABLE 5 N pg/ml IL-1βHealthy controls 13 1455 ± 585 ICA + pre-IDD 13 1189 ± 325 TNFα: Healthycontrols 10  718 ± 345 ICA + pre-IDD 14  282 ± 91

[0054] IL-10 production by monocytes of pre-IDD and healthy controlswere also examined. It was found that there was no significantdifference in the levels of IL-10 produced by either resting orstimulated monocytes from pre-IDD or healthy controls.

[0055] These data demonstrate that PGS-2 expression is not secondary tohigh levels of IL-1β or TNFα production or the lack of IL-10 secretionby pre-IDD monocytes. These findings support the notion that PGS-2expression in the pre-IDD monocyte is a primary defect.

EXAMPLE 6 PGS-2 mRNA Expression

[0056] PGS-2 mRNA expression has been found to be stable in the pre-IDDsubjects as five individuals examined on more than one occasion (usually3-6 months from the previous observation) remain positive. Likewise, sixcontrol subjects negative for PGS-2 remain negative with similarfollow-up testing (see Table 6). TABLE 6 Subject group PGS-2 +/Total %PGS-2 + Healthy controls  2/23  8.6 ICA + pre-IDD (observation) 14/1687.5* ICA + pre-IDD (insulin-treated) 15/19 73.6** ICA negative insulinautoantibody positive  2/5 40 ICA negative established IDD (>5 years) 6/7 85.7*** Systemic lupus erythematosus  5/7 71**** Autoimmunethvroiditis  2/3 66

EXAMPLE 7 PGS-2 Expression as Primary or Secondary Defect

[0057] Six long-term IDD patients (diabetes onset>5 years) and foundthat ⅚ of these individuals express PGS-2 have been analyzed. Studiesshow that ICA is lost in IDD patients within five years of the onset ofclinical diabetes, reflecting a “burned out” autoimmune process. Thesedata lend further support to the notion that PGS-2 is a primary monocytedefect in human IDD as it is in the NOD mouse.

[0058] Identification of PGS-2 as a primary defect enables the use ofPGS-2 as an early cellular marker for IDD susceptibility. Differences inthe PGS-2 gene of normal individuals and autoimmune subjects can be usedto perform genetic screening of individuals to assess susceptibility todiabetes or other autoimmune disease.

[0059] Regardless of its status as a primary defect, PGS-2 expressionreflects an active autoimmune process, and is highly advantageous inidentifying individuals at high risk for IDD. In this regard,PGS-2-positive individuals who produce the highest levels of PGS-2 haveprogressed to clinical diabetes the fastest. It is also known thatautoimmune diseases progress into spontaneous remissions orexacerbations. PGS-2 expression can be used to identify and/or monitorsuch changes in disease activity, i.e., PGS-2 positivity reflectinghigher levels of disease activity and loss of PGS-2 expressionreflecting remission. This is of great importance in IDD, where nophysical signs or symptoms manifest to suggest exacerbation of theautoimmune attack on the insulin-producing cells.

[0060] PGS-2 is expressed in a high percentage of monocytes fromindividuals with autoimmune disorders such as SLE and thyroiditis. Thus,PGS-2 expression in monocytes can be employed as a cellular marker forother autoimmune diseases in addition to IDD. Of note in screeninghealthy controls is one individual whose monocytes were stronglypositive for PGS-2 expression, who had no personal or family history ofautoimmune diseases. This individual, six weeks post-screening,developed Raynaud's phenomenon and a strongly positive ANA, suggestingthe development of a collagen vascular disease. This further supportsthe utility of the subject invention for general screening forautoimmune dysfunction.

EXAMPLE 8 FACS Assay for PGS-2

[0061] One aspect of the invention is a fluorescent activated cellsorter (FACS) based assay for PGS-2 protein. Using this assay, it ispossible to quantitate the percentage of cells in the peripheral bloodthat express the PGS-2 protein. This assay employs an antibody thatspecifically binds to the PGS-2 protein. The binding of this antibody toPGS-2 can be detected because the antibody is coupled to a fluorescentmolecule which can be detected by the lasers of the FACS machine. Usingthis procedure, which requires only one-half teaspoon of blood, it ispossible to detect the expression of PGS-2 protein in blood cells anddetermine the percentage of monocytes of pre-IDD individuals thatconstitutively express this enzyme.

EXAMPLE 9 Uses, Formulations, and Administrations

[0062] Application of the treatments of the subject invention can beaccomplished by any suitable method and technique presently orprospectively known to those skilled in the art.

[0063] In one embodiment, compounds of the subject invention haveeffective immunomodulatory activity. Specifically, they are useful inregulating immune responses in animals and humans. Thus, pharmaceuticalcompositions containing compounds of the invention as active ingredientsare useful in prophylactic or therapeutic treatment of animmunomodulatory response in humans or other mammals.

[0064] The dosage administered will be dependent upon theimmunomodulatory response desired; the type of host involved; its age,health, weight, kind of concurrent treatment, if any; frequency oftreatment; therapeutic ration and like considerations.

[0065] The compounds of the subject invention can be formulatedaccording to known methods for preparing pharmaceutically usefulcompositions. Formulations are described in detail in a number ofsources which are well known and readily available to those skilled inthe art. For example, Remington's Pharmaceutical Science by E. W. Martindescribes formulations which can be used in connection with the subjectinvention. In general, the compositions of the subject invention will beformulated such that an effective amount of the bioactive compound(s) iscombined with a suitable carrier in order to facilitate effectiveadministration of the composition.

[0066] In accordance with the invention, pharmaceutical compositionscomprising an active ingredient and one or more non-toxic,pharmaceutically acceptable carrier or diluent.

[0067] The compositions of the invention are advantageously used in avariety of forms, e.g., tablets, capsules, pills, powders, aerosols,granules, and oral solutions or suspensions and the like containingsuitable quantities of the active ingredient. Such compositions arereferred to herein and in the accompanying claims generically as“pharmaceutical compositions.” Typically, they can be in unit dosageform, namely, in physically discrete units suitable as unitary dosagesfor human or animal subjects, each unit containing a predeterminedquantity of active ingredient calculated to produce the desiredtherapeutic or prophylactic effect in association with one or morepharmaceutically acceptable other ingredients, e.g., diluent or carrier.

[0068] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and the scope of the appended claims.

I claim:
 1. A method for preventing or reducing the severity ofautoimmune disease wherein said method comprises the administration of aprostaglandin inhibitor.
 2. The method, according to claim 1 , whichcomprises the administration of a compound that decreases theproduction, or blocks the activity, of PGS-2.
 3. The method, accordingto claim 2 , wherein said compound is indomethacin.
 4. The method,according to claim 3 , which further comprises the administration ofaminoguanidine.
 5. The method, according to claim 2 , wherein saidcompound is NS-398.
 6. The method, according to claim 1 , which furthercomprises the administration of an antigen associated with saidautoimmune disease.
 7. The method, according to claim 1 , wherein saiddisease is insulin-dependent diabetes.
 8. The method, according to claim6 , wherein said antigen is selected from the group consisting ofinsulin, GAD, IA-2, and antigenic fragments and variants of theseantigens.
 9. A method for increasing the production of IL-1RA whereinsaid method comprises the administration of a PGS-2 inhibitor.